Calcific aortic valve (AV) disease is a major cause of cardiac-related deaths worldwide and is a strong risk factor for additional cardiovascular events. AV disease was originally thought to be a wear and tear process but now it is known to be an active inflammatory disease leading to sclerotic and calcific lesions. Further, AV disease occurs preferentially on the fibrosa side whereas the ventricularis side remains relatively healthy. The mechanism by which shear contributes to this disease remains unknown. We hypothesize that disturbed flow conditions are present on the fibrosa side and regulate endothelial gene expression - miRNAs and mRNAs that lead to AV inflammation and calcification. Previously, we have identified shear-responsive miRNAs in human aortic valvular endothelial cells (HAVECs); however, the importance and function of these miRNAs remains unknown. Recently, we have identified side-dependent miRNAs from freshly isolated endothelial- enriched RNA in porcine AVs. From this work, we have identified miR-199a-5p, miR-214, and miR-486-5p as the top three shear- and/or side-dependent miRNAs. We hypothesize that these are mechanosensitive miRNAs in endothelium which leads to AV inflammation and calcification. To address this hypothesis, we propose the following aims. First, we will determine the role of mechanosensitive miRNAs in aortic valve endothelium in vitro and in porcine AVs. Shear- and side-dependent expression of miR-199a-5p, 214, and 486- 5p will be validated in cultured AVECs and freshly isolated, endothelial-enriched RNA from human and porcine AVs using qPCR and in situ hybridization. The role of these miRNAs in shear-sensitive endothelial function, including inflammation, migration, apoptosis, proliferation, and cell cycle will be investigated using gain- r loss-of-function studies. Next, we will determine the role of mechanosensitive miRNAs in aortic valve endothelium ex vivo using porcine AVs. Array studies with validation will be completed in porcine AVs exposed to laminar shear or oscillatory shear stress conditions to identify shear- and side-dependent miRNAs in both endothelial-enriched RNA as well as RNA from the leftover tissue (mainly interstitial cells). Following validation, the levels of the key miRNAs (miR-199a, 214, and 486-5p) will be modulated and assessed for cell function changes pertaining to inflammation and calcification. Finally, we will determine the role of mechanosensitive miRNAs in AV calcification and inflammation in vivo. AV disease will be modeled in vivo using ApoE-/- mice fed a Western diet. Development of inflammation, sclerosis (valve thickness), apoptosis, and calcification will be characterized. This mouse model of AV calcification will be used to validate the selected miRNAs from Aims 1 and 2 in an in vivo setting with the use of LNA inhibitors or miRNA precursors. Identification of these miRNAs will provide insight to molecular mechanisms by which AV disease occurs, offering potential therapeutic targets and diagnostic markers as well as important considerations in designing tissue-engineered valves.